DOI: https://doi.org/10.52973/rcfcv-e32088
Received: 28/08/2021 Accepted: 21/02/2022 Published: 09/05/2022
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Revista Cientíca, FCV-LUZ / Vol. XXXII, rcfcv-e32088, 1 - 5
ABSTRACT
In order to determine the antibodies levels against Bovine Herpesvirus
type 1 (BHV-1) and associated risk factors in unvaccinated dairy herds
of tropical wet weather of Ecuador, an investigation was carried out
in thirteen production units (PU), in the Chone Canton, Province of
Manabí. The sample size was calculate by cluster sampling and the
variables to be evaluated were antibodies levels frequency, age, sex,
ocular or vulvovaginal lesions, breed and PU. One hundred eighty three
blood samples were taken from the jugular or caudal vein and labeled
for transfer to the laboratory. The tubes were centrifuged at 3,220
g for 15 minutes and the sera were transferred to Eppendorf tubes
that once labeled were stored at –20°C until processing. Antibodies
levels against HBV-1 were measured through of competitive ELISA
technique, and calculated with simples statistical. The risk factors
were estimated through the Odds ratio and relative risk, and the
signicance level through the Chi-square. The general antibodies
levels frequency was 58.47%. According age, 28.57% in animals
under two years old (yr.old) and 67.38% for cattle over two yr.old.
According to the sex, males showed 18.18% and females 82.61%. The
determined risk factors (P<0.05) were age and sex, with a signicant
relative risk (P<0.05) of 3.31 and 2.32, respectively. The unvaccinated
dairy herds of Western lowlands of Ecuador, have high antibodies
levels frequency against BHV-1, with females over two yr.old at the
highest risk of presenting infectious bovine rhinotracheitis (IBR).
Key words: Dairy herds; BHV-1; seroprevalence; risk factors;
unvaccinated
RESUMEN
Con el objetivo de determinar la seroprevalencia frente a Herpesvirus
Bovino tipo 1 (BHV-1) y los factores de riesgo asociados en rebaños
lecheros no vacunados de un clima húmedo tropical de Ecuador, se
llevó a cabo una investigación en 13 unidades de producción (UP), en el
cantón Chone de la provincia de Manabí. El tamaño de la muestra fue
calculado a través del muestreo por conglomerados y las variables a
ser evaluadas fueron: seroprevalencia, edad, sexo, lesiones oculares
o vulvovaginales, mestizaje y UP. Un total de 183 muestras de sangre
periférica fueron tomadas y rotuladas para su envío al laboratorio.
Los tubos fueron centrifugados a 3.220 g por 15 minutos y los sueros
transferidos a tubos Eppendorf, una vez rotulados se almacenaron a
–20°C, hasta su procesamiento. Los niveles de anticuerpos frente a
HBV-1 fueron medidos a través de la técnica de ELISA competitiva y
calculados con estadísticos simples. Los factores de riesgo fueron
estimados, a través del Odds Ratio y riesgo relativo, la signicancia
con la prueba de Ji-cuadrado. La seroprevalencia general fue 58,47%.
De acuerdo a la edad, el valor en los animales menores de dos años fue
28,57%, y en los mayores a dos años 67,38%. Los machos mostraron
un 18,18% de seroprevalencia, mientras que las hembras 82,61%.
Los factores de riesgo determinados (P<0,05), fueron la edad y el
sexo, con un riesgo relativo signicativo (P<0,05) de 3,31 y 2,32,
respectivamente. Los rebaños lecheros no vacunados evaluados,
presentan alta seroprevalencia frente a BHV-1, con hembras mayores
a dos años con mayor riesgo de presentar rinotraqueitis infecciosa
bovina (RIB).
Palabras clave: Rebaños lecheros; BHV-1; seroprevalencia; factores
de riesgo; no vacunados
Bovine Herpesvirus-1 antibodies levels and associated Risk Factors in
unvaccinated Dairy Herds from tropical wet weather, Ecuador
Niveles de anticuerpos frente a Herpesvirus Bovino tipo 1 y factores de riesgo asociados en rebaños
lecheros no vacunados en un clima húmedo tropical, Ecuador
Hitler Cedeño-Sánchez
1
, Brinner Burgos-García
1
, Jhoana Zambrano-Aveiga
1
, Marcia Jurado-Hidalgo
1
, Patricia Zambrano-Moreira
1
, María Lugo-Almarza
2
,
María Gabriela Farías
1
and Francisco Angulo-Cubillán
3,4
*
1
Universidad Laica Eloy Alfaro de Manabí, Extensión Chone, carrera de Ingeniería Agropecuaria. Chone, Manabí, Ecuador.
2
Clínica Veterinaria Bulldog. Santo
Domingo, Santo Domingo de los Tsáchilas, Ecuador.
3
Universidad UTE, Sede Santo Domingo, Facultad de Ciencias de la Salud Eugenio Espejo, carrera de
Medicina Veterinaria, Veterinary Science Research Group. Santo Domingo, Santo Domingo de los Tsáchilas, Ecuador.
4
Universidad del Zulia, Facultad de Ciencias
Veterinarias, Departamento de Sanidad Animal. Maracaibo, Zulia, Venezuela. *Email: francisco.angulo@ute.edu.ec
Bovine Herpesvirus-1 antibodies levels in unvaccinated Dairy Herds in Ecuador / Cedeño-Sanchez et al.__________________________________
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INTRODUCTION
Bovine herpesvirus type 1 (BHV-1) is a member of the Herpesviridae
family, subfamily Alphadenovirinae [1, 18, 29], which in ruminants,
domestic and wild, form a large group of pathogens with high
morbidity [20, 22]. BHV-1 is responsible for economic losses in milk
production worldwide [5, 24], due to the use of medications, milk
discarding, mortalities [3, 10, 20, 25] and reproductive failures, such
as embryonic death or abortions [6, 15], increasing the importance,
due to the lack of regularization and the globalization of animal
markets or their products [13, 16].
Only one BHV-1 serotype has been recognize, but subtypes have
been describe based on viral desoxyribonucleic acid (DNA) restriction
patterns [18]. BHV-1 is the causative agent of bovine infectious
rhinotracheitis (IBR) [26, 29], which manifests as rhinotracheitis
and conjunctivitis [28], in addition to others syndromes such as
reproductive, encephalic and enteric [4, 8, 20]. IBR is part of the bovine
respiratory disease complex, which is caused by a combination of
viral and bacterial pathogens [3]. IBR is considered a specic species
disease of cattle (Bos taurus) [26], although it has been reported that
sheep (Ovis aries) are sensitive [10], and has cross immunity with other
herpesviruses of other animals, such as goat (Capra hircus), buffalo
(Bubalus Bubalis) or cervids [5, 9, 22]. BHV-1 is found in conjunctival,
nasal and reproductive secretions [29], transmitted directly through
aerosols or by contact with infected animals, and indirectly by water
and food contaminated with body secretions, and even by semen used
in articial insemination or embryo transfer [1, 5, 24, 26].
During the pathogenesis of BHV-1 infection, the infected animal
goes through the acute or viremia phases, of latency that can generate
long-term infections, and periodic viral reactivation in the host [26,
29], where it can present sporadic periods of viral excretion and
serve as a potential source of transmission [1]. Antibodies obtained
by passive immunity through colostrum do not prevent initial viral
replication and follow the establishment of latency, becoming latent
seronegative carriers, until seroconversion or viral reactivation occurs
[14]. The main consequence of BHV-1 infection, after accessing a wide
range of organs and tissues, is to cause a variety of symptoms such
as keratoconjunctivitis, tracheitis, enteritis, infertility and abortions
[12, 15, 26].
Antibodies levels against BHV-1, has a wide range in the reported
studies of different herds, which ranges from moderate, between
20 and 30% [5, 15], to high frequency greater than 50% [1-3, 6, 12,
21, 24]. These differences between studies could be due to types of
productive systems [20, 24], preventive measures used [1, 24], or the
viral isolate involved [20]. Among the factors that have been reported
as risk, there is mainly age, where older animals are the ones with
the highest seroprevalence [6, 7, 12, 24, 29], sex [1, 24], the history of
reproductive problems in herds [12, 20], and the non-implementation
of preventive measures [14, 15, 18, 20, 28, 29]. BHV-1 antibodies levels
frequency in Ecuador was established at 43% [7], but additional
studies have not been reported and the current status of the disease
is unknown. In the Ecuador Coast Region were used largely for cattle
meat and milk production, where one of the main production areas
is the Chone Canton of the Province of Manabí, being its economic
axis. Cattle production is mostly traditional, where preventive
measures such as vaccination of different diseases, including IBR,
are not carried out. For this reason, an investigation was carried
out to determine seroprevalence against BHV-1 and associated risk
factors, in unvaccinated herds of the tropical wet weather of Ecuador.
MATERIALS AND METHODS
Ethical considerations
To carry out this research, the Norwegian National Research
Ethics Committees guidelines for the use of animal in research were
considered [17].
Study location
The research work was carried out in the Convent Parish, in the
Chone Canton in the Province of Manabí, Ecuador. Geographically
it is between 0°11'35.31" South and 79°54'01.60" West, at 296 meters
above sea level and an average annual temperature ranging from
23 to 30°C. The climate is humid tropical with an annual average of
1,240 cubic millimeters (mm) [11]. It is an area of cattle production,
with extensive grazing in grasslands of introduced pastures (Panicum
maximun, Brachiaria spp.), with sources of water from springs, rivers,
dams and underground wells, with low use of technologies and no
history of prevention against IBR.
Study design
Thirteen dairy PU were used, which had 1,010 animals over six
month’s (mos) olds, at study time [20, 24]. The size of the sample was
determined by cluster sampling, where it is calculated by combining all
the animals of herds under study, using the probability proportional to
the size of each herd [30], and the cattle to be sampled were randomly
selected according to age group in proportion to percentage of different
age groups within each herd [27]. The dependent variable studied
was antibodies levels against BHV-1 frequency and the independents
were age (greater than two yr., less than two yr.), sex (male, female),
presence of vulvovaginal or ocular lesions, breed according to their
phenotypic characteristics (Bos taurus taurus and B. t. indicus) and PU.
Population sample size
The sample size of the bovine population of the 13 PU was 183
animals, of which 141 were older than two yr old. and 42 were younger,
22 males and 141 females, 107 had vulvovaginal or ocular lesions, and
the animals’ number according to their racial predominance was: 98
B. t. indicus and 85 B. t. taurus, respectively.
Blood serum sample collection and storage
Blood sampling was collected via jugular vein in the case of young
animals, and the caudal vein in adult, using Vacutainer® system. The
samples were labeled and transferred in refrigeration in cellar at 4°C
(Ecoshel, C800D, Mexico), to animal health laboratory of the Universidad
Eloy Alfaro de Manabí Extension Chone. The samples were centrifuged
at 3,220 g for 15 minutes (min), for blood serum extraction, which was
deposited in Eppendorf® tubes, identied and stored at –20°C (Thermo
Scientic, 25LCETSSA, USA) until its processing [29].
Competitive Elisa technique
For serological diagnosis, the sera were thawed at room temperature
and analyzed using the competitive ELISA technique. The entire
technique procedure was performed following the instructions of
the manufacturer’s protocol. The ID Screen® IBR gB competition Kit
from the ID laboratory was used (Vet Innovative Diagnostics, Grabels-
FRANCE), for the detection of anti-gB BHV-1 antibodies in serum
samples [19].
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Antibodies level was calculated according to the percentage of
competition (S/N %), which was calculated using the following formula
with optical density (OD) data:
/%SN
Od Negativecontrol
OD simple
100
#
=
Where, if the result was less than or equal to 45%, they are considered
positive, greater than 45% and less than 55%, they are considered
doubtful, and greater than or equal to 55%, they are considered
negative [19]. Once the positive and negative animals were obtained,
the seroprevalence was calculated with epidemiological formulas.
Analysis of data
BHV-1 antibodies levels were analyzed through simples statistical
and the risk factors determination for the independent variables was
obtained through of Odds ratio and relative risk calculation [31], and
the degree of signicance was estimated by the Chi-square test,
through the Statistical Analysis System (SAS) [23].
RESULTS AND DISCUSSION
The results of seroprevalence against BHV-1, in dairy herds of the
western lowlands of Ecuador, show a high overall seroprevalence
in the herds studied, with 107 positive animals, 74 negative and two
doubtful, for 58.47%, as shown in TABLE I.
The seroprevalence against BHV-1 in unvaccinated cattle herds,
in western lowlands of Ecuador was determined and had a high
value of positive animals with 58.47%. This value was like that
reported in other studies, including unvaccinated herds [1], which
showed high seroprevalences, above 50% [2, 3, 6, 12, 21, 24]. The
high number of positive animals may be related to different factors,
including the isolate involved [1], the ability of the virus to remain
latent, the introduction of infected animals that spread disseminate
and perpetuate the virus within the herds [9, 29] and the control
measures implemented [15]. Also suggests a viral free circulation
in the environment, with continuous or periodic excretions of the
infectious agent by latent infected animals or with viral reconversion
of the herds [1], which suggests the endemic presence of IBR [15].
The entry of BHV-1 in the herds, can be given in the incorporation
of animals, both heifers and bulls [9, 12, 29], that latter copulate
with many females, causing rapid viral dissemination, when natural
mountaineering is used, although through articial insemination,
transmission could also occur, because semen has been shown to
be an important route of excretion of BHV-1 [9].
Among the risk factors studied, age and sex were variables that
increased the relative risk of nding animals infected with BHV-1. In
the case of age, it has been reported by different authors that the
adults’ animals have highest risk of being infected [24, 29]. Antibodies
against the virus in calves under six mos old of age, probably
correspond to passive maternal immunity from colostrum, which is
lost around the yr of life, starting the increase in serological prole
against BHV-1 in the second yr., due to active infection [29]. Likewise,
lasting viral exposure by adult animals increases seroprevalence [1],
without ruling out the additive effect of reinfections and the fact
that adults are in reproductive age, increasing the risk of acquire
the infection through natural mounts or articial insemination [9].
TABLE I
General BHV-1 antibodies
frequency in the herds studied
Results n (%)
Positive 107 58.47
Negative 74 40.44
Suspects 2 1.09
Total 183 100.00
In TABLE II, seroprevalence can be observed according to the
sampled animals’ age, in addition to the value of Odds ratio and relative
risk. Seroprevalence was lower for animals younger than two yr-old
with 28.57% and higher in those older than two yr-old with 67.38%,
determining age as a risk factor, showing a relative risk value of 3.31
times higher in adult animals to presence of seropositivity, compared
to young animals. Likewise, according to sex, the results show a
seroprevalence for males of 18.18 and 82.61% for females, this variable
being a risk factor (P<0.05), since females showed 2.32 times more
risk of being seropositive to BHV-1, compared to males (TABLE III).
The seroprevalence according to the presence of vulvovaginal
or ocular lesions was 40.19%, with a relative risk of 1.05 (P>0.05).
Likewise, according to the breed predominance (p), the B. p. indicus
52.55% and B. p. taurus 51.23%, respectively, with a relative risk of
1.29 (P<0.05). In the case of PU, 100% showed animals seropositive
to HBV-1.
TABLE II
BHV-1 antibodies frequency according to
Age, Odds ratio, Relative Risk
Age n Positives (%) Odds ratio Relative Risk
< 2 years 42 12 28.57
> 2 years 141 95 67.38 5.02* 3.31*
Total 183 107 58.47
*P Value < 0.05.
TABLE III
BHV-1 antibodies levels frequency according
to Sex, Odds ratio, Relative Risk
Sex n Positives (%) Odds ratio Relative Risk
Males 22 02 18.18
Females 161 133 82.61 4.64* 2.32*
Total
183 107 58.47
*P Value < 0.05.
Bovine Herpesvirus-1 antibodies levels in unvaccinated Dairy Herds in Ecuador / Cedeño-Sanchez et al.__________________________________
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Sex increased the relative risk of acquiring the infection, with females
more likely than males, as was shown by Saravanajayam et al. [24].
These results suggest that females are mostly exposed to the virus,
since under traditional conditions of bovine exploitation, there is a
male: female ratio of 1:25, which suggests greater contact between
an infected bull with several cows, relative to a cow infected with the
rest of its herd. The presence of ocular or vulvovaginal lesions was
not related to seroprevalence, which may be due to the fact that the
circulating virus is of low virulence [3].
CONCLUSION
The unvaccinated bovine herds of tropical wet weather of Ecuador,
have high seroprevalence against BHV-1, with females over two
years old of age with highest risk of presenting serum antibodies.
Vulvovaginal or ocular lesions, breed and PU, were not determinants
when assessing the risk factors on the seroprevalence of BHV-1
infection.
ACKNOWLEDGMENTS
The authors wish to thank the Universidad Laica Eloy Alfaro de
Manabí – Extension Chone, Universidad UTE – Sede Santo Domingo,
and Universidad del Zulia, for the co-nancing of this research.
CONFLICT OF INTEREST
The authors declare that they have no conict of interest.
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